VHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cross-seeding on the stability of fibrils

The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty amyloidoses are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1]. Although the mechanism of amyloid fibril formation at the molecular level is not yet completely understood, it has been shown that the capacity to form amyloid fibrils in vitro is an intrinsic property of all polypeptide chains [1]. The choice of model proteins to investigate the aggregation process in vitro is therefore not restrained to proteins involved in amyloidoses but can be settled on a wide variety of proteins.
In this study, we have chosen to investigate the mechanism of amyloid fibril formation by two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, for which variants with mutations located at the disulfide bond [3,4] and the CDRs [3] are available. Characterisation of the aggregating properties of these mutants will allow the investigation of the impact of these structural elements on the process of fibril formation.
In order to determine conditions in which cAb-HuL6 and cAb-BcII10 are more susceptible to form amyloid fibrils, heat-induced unfolding experiments at several pHs have been monitored by intrinsic fluorescence and circular dichroism. Then, aggregation experiments have been performed in the selected conditions and the presence of amyloid fibrils has been acknowledged by thioflavineT fluorescence experiments and electron microscopy. We will discuss the kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding and the stability of the formed fibrils.

[1] Chiti and Dobson, Annu. Rev. Biochem., 75, 2006, 333-366 ; [2] Dumoulin et al., Protein Sci., 11, 2002, 500-515 ; [3] Saerens et al., J. Mol. Biol., 352, 2005, 597-607 ; [4] Saerens et al., J. Mol. Biol., 377, 2008, 478-488.